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Abstract

Background

Despite years of effort, a licensed malaria vaccine is not yet available. One of the
obstacles facing the development of a malaria vaccine is the extensive heterogeneity
of many of the current malaria vaccine antigens. To counteract this antigenic diversity,
an effective malaria vaccine may need to elicit an immune response against multiple
malaria antigens, thereby limiting the negative impact of variability in any one antigen.
Since most of the malaria vaccine antigens that have been evaluated in people have
not elicited a protective immune response, there is a need to identify additional
protective antigens. In this study, the efficacy of three pre-erythrocytic stage malaria
antigens was evaluated in a Plasmodium yoelii/mouse protection model.

Methods

Mice were immunized with plasmid DNA and vaccinia virus vectors that expressed one,
two or all three P. yoelii vaccine antigens. The immunized mice were challenged with 300 P. yoelii sporozoites and evaluated for subsequent infection.

Results

Vaccines that expressed any one of the three antigens did not protect a high percentage
of mice against a P. yoelii challenge. However, vaccines that expressed all three antigens protected a higher
percentage of mice than a vaccine that expressed PyCSP, the most efficacious malaria
vaccine antigen. Dissection of the multi-antigen vaccine indicated that protection
was primarily associated with two of the three P. yoelii antigens. The protection elicited by a vaccine expressing these two antigens exceeded
the sum of the protection elicited by the single antigen vaccines, suggesting a potential
synergistic interaction.

Conclusions

This work identifies two promising malaria vaccine antigen candidates and suggests
that a multi-antigen vaccine may be more efficacious than a single antigen vaccine.

Background

Malaria kills approximately 863,000 people every year [1]. Although a variety of anti-malarial drugs exist, the cost of these drugs can be
prohibitive in the relatively poor areas of the world where malaria is endemic. The
wide-spread use of the most commonly employed drugs has also resulted in the expansion
of drug-resistant parasites, rendering many of these drugs ineffective [2]. In the absence of inexpensive, highly potent drugs, vaccination represents the most
cost-effective way of supplementing traditional malaria interventions.

A successful malaria vaccine will need to protect people against a large population
of antigenically diverse malaria parasites. A vaccine based on a single isolate of
a single antigen may not be able to elicit an immune response that is broad enough
to protect individuals against this heterogeneous population. Therefore, an efficacious
malaria vaccine may need to induce an immune response against multiple malaria antigens,
a belief that has propelled the development of whole-organism malaria vaccines, such
as the irradiated sporozoite vaccine and the genetically attenuated sporozoite vaccine
[3,4].

A variety of malaria vaccine candidates are being evaluated in clinical trials throughout
the world. The most advanced vaccine candidate, RTS,S, is currently being evaluated
in a phase 3 trial at 11 sites in seven African countries. RTS,S is a recombinant
protein vaccine based on the Plasmodium falciparum circumsporozoite protein (CSP). It has protected malaria-naïve adults against an experimental
P. falciparum challenge and reduced malaria-associated episodes in children living in malaria endemic
areas [5,6]. The level and duration of immunity induced by RTS,S, however, is relatively modest.

One way to potentially enhance the efficacy of RTS,S, or any other subunit malaria
vaccine, would be to incorporate additional malaria antigens into the vaccine, thereby
broadening the immune response elicited by the vaccine. At least one other malaria
antigen has protected volunteers against a malaria challenge. A prime-boost regimen
with adenovirus and poxvirus vectors expressing P. falciparum thrombospondin-related adhesive protein (TRAP) has protected volunteers against an
experimental P. falciparum challenge [7]. A prime-boost regimen with DNA and adenovirus vectors expressing CSP and apical
membrane antigen 1 (AMA1) has also protected volunteers against an experimental P. falciparum challenge [8]. Although the data from both of these clinical trials are not yet published, these
studies indicate that CSP, TRAP and possibly AMA1 can induce protective immune responses
in people. Unfortunately, most of the other malaria vaccine antigens evaluated in
people have not induced significant levels of protection. For example, recombinant
protein vaccines containing the C-terminal end of the merozoite surface protein 1
(MSP142), the AMA1 ectodomain or a combination of three P. falciparum antigens (MSP1, MSP2 and ring-infected erythrocyte surface antigen (RESA)) have not
induced significant levels of protection against natural infection in children living
in malaria endemic regions [9-11]. Each of these vaccines, however, may have induced some level of strain-specific
protection against the P. falciparum strain from which the vaccine antigen was derived [11,12]. Since an immune response against multiple malaria antigens may be necessary to protect
a high percentage of people against the large number of antigenically diverse P. falciparum strains throughout the world, there is a great need to identify new malaria vaccine
antigens.

In this report, the efficacy of three malaria vaccine antigens was evaluated in a
P. yoelii/mouse model. Although these three pre-erythrocytic stage antigens, PY03011, PY03424
and PY03661, were independently identified by our bioinformatic and genomic analyses,
two of the antigens (or their orthologs) were previously described (PY03011 = PyUIS3,
PY03424 = falstatin) [13,14]. Protection studies with DNA and vaccinia virus vaccine vectors expressing these
antigens suggest that two of the antigens, PY03011 and PY03424, can protect mice against
a P. yoelii sporozoite challenge.

Methods

Down-selection of vaccine candidate genes

P. falciparum and P. yoelii express approximately 5,800 genes. It is not feasible to evaluate the vaccine potential
of that many genes. Therefore, various methods were used to down-select the most promising
vaccine candidates. Assuming that a vaccine based on a pre-erythrocytic antigen is
more likely to be successful than a vaccine based on an erythrocytic antigen, the
down-selection process focused on sporozoite and liver stage antigens. To identify
promising sporozoite antigens, genomic and proteomic information contained in pre-existing
malaria databases was evaluated [15,16]. To identify promising liver stage antigens, an expression library created with material
isolated from P. yoelii-infected liver cells was evaluated [17]. The P. falciparum genes encoding the down-selected sporozoite and liver stage antigens were cloned using
a high-throughput cloning strategy [18]. Evaluation of the proteins encoded by these genes with antisera from volunteers
who had received a P. falciparum irradiated sporozoite vaccine identified 20 promising vaccine candidates [19].

Generation of DNA and vaccinia virus vaccine vectors

The re-annotated single exon PY03011 gene was isolated from P. yoelii (17XNL) genomic DNA by PCR with the primers, 5'-TGGATCCATGAAAGTGTATAAAATGAACACTCTC-3'
and 5'-TGGATCCTCATTTTGGTTGATATTGTTCTTTAAG-3'. The DNA-PY03011 vaccine vector was generated
by cloning the PY03011 gene from this PCR reaction into the BamHI site of the DNA vaccine vector, VR1020 (Vical Inc., San Diego, CA). This cloning
reaction positions the full length PY03011 gene downstream from a cytomegalovirus
(CMV) immediate-early (IE) promoter and in-frame with a human tissue plasminogen activator
(TPA) signal sequence. Since the PY03011 protein contains a signal sequence, cloning
the PY03011 gene into VR1020 downstream from an in-frame TPA signal sequence results
in a PY03011 construct that contains two signal sequences. The vaccinia-PY03011 vaccine
vector was generated using a host range selection system [20]. The full length PY03011 gene in this vector is inserted into the vaccinia virus
A-type inclusion body (ATI) locus and is under the transcriptional control of a synthetic
early/late (E/L) promoter [21].

Exon 2 of the re-annotated PY03424 gene was isolated from P. yoelii (17XNL) genomic DNA by PCR with the primers, 5'-TGGATCCTACTCTTTTGACATTGTAAACGAG-3'
and 5'-TGGATCCTTATTGGACAGTTACGTATAAAATTTTAG-3'. The DNA-PY03424 vaccine vector and
the vaccinia-PY03424 vaccine vector were generated with the same reagents and techniques
used to generate the DNA-PY03011 and vaccinia-PY03011 vectors. The DNA and vaccinia
vaccine vectors expressing PY03424 (exon 2) do not contain the first 26 codons of
the PY03424 gene. Since the first 21 codons of the PY03424 gene encode a signal sequence,
the PY03424 proteins expressed by the DNA-PY03424 and vaccinia-PY03424 vectors do
not contain the native PY03424 signal sequence. To enhance expression, the DNA-PY03424
vector was engineered to express a PY03424 protein with a TPA signal sequence and
the vaccinia-PY03424 vector was engineered to express a PY03424 protein with a human
decay accelerating factor (DAF) signal sequence.

The PY03661 gene was isolated from P. yoelii (17XNL) genomic DNA by PCR with the primers, 5'-TGGATCCATGTTTCGATCTGATTCCCATTTCC-3'
and 5'-TGGATCCTTATGTTTGATGATAATTTTCTTTCG-3'. The DNA-PY03661 vaccine vector and the
vaccinia-PY03661 vaccine vector were generated with the same reagents and techniques
used to generate the other DNA-P. yoelii and vaccinia-P. yoelii vectors. Since the native PY03661 gene does not contain a signal sequence, the vaccinia-PY03661
expression cassette was constructed with a DAF signal sequence. Therefore, the DNA-PY03661
vector expresses a PY03661 protein with a TPA signal sequence and the vaccinia-PY03661
vector expresses a PY03661 protein with a DAF signal sequence.

Female CD1 mice were injected subcutaneously in the tail and scruff of the neck on
days 0 and 29 with 10 μg of PY03661, PY03424 (exon 2) or PY03661 recombinant protein
adjuvanted in Montanide™ ISA720. On day 38, the mice were bled and P. yoelii protein-specific antisera prepared.

Indirect fluorescent antibody analyses

Indirect fluorescent antibody (IFA) assays with sporozoite and blood stage parasites
were performed as previously described [24]. In brief, serial dilutions of P. yoelii protein-specific antisera were incubated with P. yoelii (17XNL) sporozoites or blood from P. yoelii-infected mice. Parasites were visualized with a fluorescein isothiocyanate (FITC)
conjugated goat anti-mouse IgG (KPL Inc., Gaithersburg, MD). IFA analyses with liver
stage parasites were performed as previously described [25]. In brief, mice were infected with P. yoelii sporozoites and livers were harvested 48 hours post-infection. P. yoelii-infected liver sections were prepared and incubated with P. yoelii protein-specific antisera. Parasites were visualized with a FITC conjugated goat anti-mouse
IgG. Evans blue (0.02%) counterstain was added to the secondary antibody, providing
a red background to contrast the green FITC fluorescence when excited at the same
wavelength.

Protection studies

Female CD1 mice were injected intramuscularly in the tibialis anterior muscle with
100 μl of vaccine (50 μl in each leg) using a 0.3 ml syringe and a 29G1/2 needle (Becton
Dickinson Co., Franklin Lakes, NJ) fitted with a plastic collar cut from a micropipette
tip [26]. The DNA vaccine vectors were prepared in 1X Phosphate Buffered Saline (PBS) and
diluted to the appropriate concentration for vaccination in 1X PBS. The vaccinia vaccine
vectors were prepared in 1 mM Tris (9.0) and diluted to the appropriate concentration
for vaccination in 1X PBS. Mice were challenged intravenously in the tail vein with
300 P. yoelii (17XNL) sporozoites using a 1 ml syringe and 26G1/2 needle (Becton Dickinson Co.,
Franklin Lakes, NJ). Sporozoites were hand dissected from infected mosquito salivary
glands and diluted for challenge in M199 medium containing 5% normal mouse serum (Gemini
Bio-Products, West Sacramento, CA).

In protection study 1, 14 mice per group were primed on day 0 with 100 μg of the appropriate
DNA-P. yoelii vaccine vector and boosted on day 40 with 5 × 107 plaque forming units (pfu) of the corresponding vaccinia-P. yoelii vaccine vector. Mice immunized with a combination of vectors expressing PY03011, PY03424
and PY03661 were primed with a total of 300 μg of the DNA-P. yoelii vectors and boosted with a total of 1.5 × 108 pfu of the vaccinia-P. yoelii vectors. Vaccine vectors expressing PyCSP were included in each study as a positive
control. On day 50, the mice were bled and sera prepared. On day 54, the mice were
challenged with 300 P. yoelii sporozoites. On days 61-68, parasitaemia was evaluated by examining Giemsa-stained
blood smears. Mice were considered positive if parasites were observed in any sample.
To gauge the severity of the challenge, four groups of naïve CD1 mice were challenged
with four suboptimal doses of P. yoelii sporozoites (calculated through serial dilution to be approximately 100, 33.3, 11.1
or 3.7 sporozoites per mouse). From these infectivity control mice, an ID50 was calculated. (An ID50, or infectious dose 50, equals the dose of sporozoites required to infect 50% of
the mice.) Extrapolation from these results indicated that the mice injected with
300 P. yoelii sporozoites were challenged with a dose equivalent to seven times the ID50 dose.

In protection study 2, 14 mice per group were primed on day 0 with 100 μg of the appropriate
DNA-P. yoelii vaccine vector and 30 μg of a DNA vector expressing murine granulocyte-macrophage
colony-stimulating factor (mGM-CSF) and boosted on day 42 with 3.3 × 107 pfu of the corresponding vaccinia-P. yoelii vaccine vector. Mice immunized with two or three DNA-P. yoelii vectors were primed with a total of 200 μg or 300 μg of the DNA-P. yoelii vectors and 30 μg of the DNA-mGM-CSF vector and boosted with a total of 6.6 × 107 pfu or 1 × 108 pfu of the vaccinia-P. yoelii vectors. Three separate groups of negative control mice were immunized with three
different doses of DNA and vaccinia vectors that do not express a P. yoelii antigen. One group was primed with 100 μg of an "empty" DNA vector and 30 μg of a
DNA-mGM-CSF vector and boosted with 3.3 × 107 pfu of an "empty" vaccinia vector. A second group was primed with 200 μg of an "empty"
DNA vector and 30 μg of a DNA-mGM-CSF vector and boosted with 6.6 × 107 pfu of an "empty" vaccinia vector. A third group was primed with 300 μg of an "empty"
DNA vector and 30 μg of a DNA-mGM-CSF vector and boosted with 1 × 108 pfu of an "empty" vaccinia vector. On day 52, the mice were bled and sera prepared.
On day 57, the mice were challenged with 300 P. yoelii sporozoites. On days 64-71, parasitaemia was evaluated by examining Giemsa-stained
blood smears. Mice were considered positive if parasites were observed in any sample.
To gauge the severity of the challenge, four groups of naive mice were challenged
with four suboptimal doses of P. yoelii sporozoites (100, 33.3, 11.1 or 3.7 sporozoites). From these infectivity control mice,
it was calculated that the mice injected with 300 P. yoelii sporozoites in this study were challenged with a dose equivalent to 13.6 times the
ID50 dose.

The regimens for the two protection studies were slightly different. For example,
the dose of the individual vaccinia-P. yoelii vectors was slightly higher in protection study 1 (5 × 107 pfu) than protection study 2 (3.3 × 107 pfu). Consequently, the total dose of the trivalent vaccine was 1.5 × 108 pfu in protection study 1 and 1 × 108 pfu in protection study 2. Additionally, in protection study 2, the DNA vectors were
mixed with a DNA-mGM-CSF plasmid. Although previous studies had indicated that co-administration
of a DNA-PyCSP vector with a DNA-mGM-CSF plasmid could enhance the immunogenicity
and efficacy of a DNA-vaccinia prime-boost regimen [27], this enhancement is greater in inbred mouse strains (BALB/c and C57BL/6) than outbred
strains [28]. Therefore, it is not surprising that the DNA-mGM-CSF plasmid did not appear to enhance
the efficacy of the PyCSP or trivalent P. yoelii vaccines in protection study 2, relative to protection study 1.

Statistical analyses

Results

Genomic characterization of three P. yoelii vaccine antigens

Analysis of pre-erythrocytic P. falciparum proteins with sera from human volunteers immunized with a P. falciparum irradiated sporozoite vaccine identified 20 promising vaccine antigens [19]. To evaluate the vaccine potential of these proteins in a murine protection model,
vaccine vectors that express the P. yoelii ortholog of three of these antigens, PY03011, PY03424 and PY03661, were generated.

PY03011

PY03011 is predicted by PlasmoDB [29], a Plasmodium database, to be the ortholog of the P. falciparum gene, PF13_0012. PF13_0012 is predicted by PlasmoDB to be a single exon gene that
encodes a protein that is 229 amino acids long. PY03011 is predicted by PlasmoDB to
contain two exons and encode a protein that is 241 amino acids long. The first exon
is predicted to encode the first 16 amino acids and the second exon is predicted to
encode the remaining 225 amino acids. A previous study, however, annotated the PY03011
gene to be a single exon gene that encodes a protein that is 220 amino acids long
[30]. The re-annotated PY03011 protein is more homologous to PF13_0012 than the PlasmoDB-annotated
PY03011 protein (30% vs. 28%). The single exon annotation is also consistent with
the annotations of the P. falciparum, Plasmodium berghei, Plasmodium chabaudi, Plasmodium knowlesi and Plasmodium vivax orthologs of this gene, which are predicted by PlasmoDB to be single exon genes. Based
upon these data, the studies in this report were performed with a single exon PY03011
gene that encodes a protein that is 220 amino acids long [30].

The re-annotated PY03011 protein is predicted to contain a signal sequence with a
cleavage site between amino acids 30-31 and a transmembrane domain between amino acids
59-81. IFA analyses with PY03011-specific antisera indicate that PY03011 is expressed
in the sporozoite, but not in the liver or blood stages of the P. yoelii life-cycle (Figure 1). A previous study indicated that this protein was expressed in the sporozoite and
liver stages [13]. Therefore, it is likely that PY03011 is expressed in the liver, but at levels that
are below the level of detection with the serological reagents used in the present
study. The genetic characteristics of the re-annotated PY03011 gene are summarized
in Table 1.

PY03424

PY03424 is predicted by PlasmoDB to be the ortholog of the P. falciparum gene, PFI0580c. PFI0580c is predicted by PlasmoDB to contain two exons and encode
a protein that is 413 amino acids long. The first exon is predicted to encode the
first 22 amino acids and the second exon is predicted to encode the remaining 391
amino acids. PY03424 is predicted by PlasmoDB to contain two exons and encode a protein
that is 1,856 amino acids long. The first exon is predicted to encode the first 1,521
amino acids and the second exon is predicted to encode the remaining 335 amino acids.
We believe that PY03424 is not annotated correctly and have re-annotated this gene.
The re-annotated PY03424 gene is predicted to contain two exons and encode a protein
that is 357 amino acids long. The first exon of the re-annotated gene encodes 22 amino
acids and the second exon encodes the remaining 335 amino acids. The re-annotated
PY03424 protein is significantly more homologous to PFI0580c than the PlasmoDB-annotated
PY03424 protein (33% vs. 6%). A comparison of PY03424 with other Plasmodium orthologs also suggests that the PlasmoDB annotation is not correct.

The re-annotated PY03424 protein is predicted to contain a signal sequence with a
cleavage site between amino acids 21-22. IFA analyses with PY03424-specific antisera
indicate that PY03424 is expressed in sporozoites, on the parasitophorous vacuole
of the liver stage and in the blood stage (Figure 1). This profile is similar to the expression profile of the P. falciparum PFI0580c ortholog, which is expressed in the sporozoite, liver and blood stages of
the P. falciparum life-cycle [14,15,19]. The genetic characteristics of the re-annotated PY03424 gene are summarized in Table
1.

PY03661

PY03661 is predicted by PlasmoDB to be the ortholog of the P. falciparum gene, PFC0555c. PFC0555c is predicted by PlasmoDB to be a single exon gene and encode
a protein that is 233 amino acids long. PY03661 is predicted to be a single exon gene
and encode a protein that is 225 amino acids long. The homology between the PFC0555c
and PY03661 proteins is 60%. PY03661 does not appear to contain a signal sequence
or a transmembrane domain. IFA analyses with PY03661-specific antisera indicate that
PY03661 is expressed in sporozoites, but not in the liver or blood stages (Figure
1). The P. falciparum PFC0555c ortholog is expressed in the sporozoite and liver stages of the P. falciparum life-cycle [19]. Since PFC0555c is expressed in the liver stage, it is likely that PY03661 is also
expressed in the liver, but at levels that are below the level of detection with the
serological reagents used in this study. The genetic characteristics of PY03661 are
summarized in Table 1.

Protection studies in P. yoelii/mouse model

To evaluate the vaccine potential of the three P. yoelii antigens, CD1 outbred mice were immunized in a heterologous prime-boost regimen with
DNA and vaccinia virus vectors that express PY03011, PY03424 or PY03661, or a combination
of these vectors (Table 2). As a positive control, mice were immunized with DNA and vaccinia vectors that express
P. yoelii CSP (PyCSP). As a negative control, mice were immunized with "empty" DNA and vaccinia
vectors that do not express a P. yoelii protein. Two weeks after the vaccinia vector boost, the mice were challenged with
300 P. yoelii sporozoites. Seven through fourteen days after the challenge, protection against blood
stage parasitaemia was evaluated by examining Giemsa-stained blood smears. None of
the 14 mice immunized with vectors that express PY03011 or PY03661 were sterilely
protected (0% protection) and only two of 14 mice immunized with vectors that express
PY03424 were sterilely protected (14% protection). However, eight of 14 mice immunized
with all three antigens were sterilely protected (57% protection) (Figure 4). The protection elicited by these three P. yoelii antigens was greater than the protection elicited by PyCSP (57% vs. 36%).

Figure 4.Protection study 1. Fourteen CD1 outbred mice per group were immunized in a prime-boost regimen with
DNA and vaccinia vectors that express PY03011, PY03424 or PY03661, or a combination
of vectors that express all three P. yoelii antigens. Positive control mice were immunized with DNA and vaccinia vectors that
express PyCSP. Negative control mice were immunized with DNA and vaccinia vectors
that do not express a P. yoelii antigen. Groups are designated (dose = 1X) or (dose = 3X) to represent the relative
quantity of the DNA and vaccinia vectors that they received. The mice were challenged
with 300 P. yoelii sporozoites and evaluated for parasitaemia by examining Giemsa-stained blood smears.

To confirm these results and determine which combination of antigens was responsible
for protection, a second efficacy study was performed. CD1 outbred mice were immunized
with DNA and vaccinia virus vectors that express PY03011, PY03424 or PY03661 (Table
2). In this study, however, separate groups of mice were immunized with a combination
of vectors that express PY03011 and PY03424, or PY03424 and PY03661, or all three
P. yoelii antigens. The PY03011 and PY03661 combination was not tested since previous studies
had suggested that PY03424 was the primary protective antigen. As a positive control,
mice were immunized with DNA and vaccinia vectors that express PyCSP. Since the mice
immunized with multiple vectors received two or three times more vaccine than the
mice immunized with a single vector, three separate groups of negative control mice
were immunized with the same amount of "empty" DNA and vaccinia vectors as the mice
that received either one, two or three vaccine vectors. Two weeks after the vaccinia
vector boost, the mice were challenged with 300 P. yoelii sporozoites. Seven through fourteen days after the challenge, protection against blood
stage parasitaemia was evaluated by examining Giemsa-stained blood smears. None of
the mice immunized with vectors that express PY03661 were protected (0% protection)
and only one of 14 mice immunized with vectors that express PY03011 or PY03424 were
protected (7% protection). However, six of 14 mice immunized with PY03011 and PY03424
were protected (43% protection), three of 14 mice immunized with PY03424 and PY03661
were protected (21% protection) and six of 14 mice immunized with all three P. yoelii antigens were protected (43% protection) (Figure 5). The protection elicited by PY03011 and PY03424 is statistically significant (PY03011/PY03424
(dose = 2X) vs. Neg con (dose = 2X), p = 0.0159). Similar to the previous study, the
protection elicited by the combination of PY03011 and PY03424 or all three antigens
was greater than the protection elicited by PyCSP (43% vs. 14%).

Figure 5.Protection study 2. Fourteen CD1 outbred mice per group were immunized in a prime-boost regimen with
DNA and vaccinia vectors that express PY03011, PY03424 or PY03661, or a combination
of vectors that express two or all three P. yoelii antigens. Positive control mice were immunized with DNA and vaccinia vectors that
express PyCSP. Three separate groups of negative control mice were immunized with
three different doses of DNA and vaccinia vectors that do not express P. yoelii antigens. Groups are designated (dose = 1X), (dose = 2X) or (dose = 3X) to represent
the relative quantity of the DNA and vaccinia vectors that they received. The mice
were challenged with 300 P. yoelii sporozoites and evaluated for parasitaemia by examining Giemsa-stained blood smears.

Discussion

In this report, the efficacy of three pre-erythrocytic stage malaria antigens, PY03011,
PY03424 and PY03661, was evaluated. DNA and vaccinia virus vectors expressing these
three antigens were evaluated in two P. yoelii protection studies. In the first study, a trivalent vaccine that expressed all three
antigens protected a significantly higher percentage of mice than vaccines that expressed
either antigen alone. Since the percentage of mice protected by the trivalent vaccine
(57%) exceeded the sum of the percentages protected by the univalent vaccines (14%),
these results suggest a potential synergistic interaction. In the second study, a
bivalent vaccine that expressed PY03011 and PY03424 protected an equivalent percentage
of mice as the trivalent vaccine, suggesting that PY03011 and PY03424 are the primary
antigens responsible for protection. A bivalent vaccine that expressed PY03424 and
PY03661 also protected a higher percentage of mice than either vaccine alone. However,
the level of protection induced by this bivalent vaccine was not statistically significant
relative to the PY03424, PY03661 or negative control groups. Similar to the first
study, the number of mice protected by the trivalent vaccine (43%) or the PY03011
and PY03424 bivalent vaccine (43%) was larger than the sum protected by the univalent
vaccines (14%), a result consistent with synergistic protection. These studies indicate
that PY03011 and PY03424, and their P. falciparum orthologs, are potential malaria vaccine antigens.

PY03011 and PY03424, and/or their P. falciparum or P. berghei orthologs, have been previously characterized. PY03011, and its P. berghei ortholog, were initially identified by differential stage-specific expression studies,
which resulted in it being designated PyUIS3 (upregulated in infectious sporozoites
3) [31,32]. Further studies indicated that PyUIS3/PY03011 is expressed in the liver stage parasitophorous
vacuole, where it binds to the host cell fatty acid carrier, liver-fatty acid binding
protein (L-FABP), and facilitates the importation of fatty acids from the hepatocyte
to the parasite [13]. Down-regulation of L-FABP inhibits parasite growth. Therefore, although the parasite
can synthesize fatty acids, it appears that it supplements this endogenous fatty acid
production by importing fatty acids from the host [13]. PyUIS3/PY03011 is homologous with a family of Plasmodium proteins called early transcribed membrane proteins (ETRAMPs). All of the ETRAMPs
share a similar structure; an N-terminal signal sequence followed by a short lysine-rich
region, a second transmembrane domain and a C-terminal region of variable length [33]. Like PyUIS3/PY03011, ETRAMPs have been shown to localize to the parasitophorous
vacuole. Unlike PyUIS3/PY03011, which is expressed in the sporozoite and liver stages,
many of the ETRAMPs are expressed exclusively in the ring stage. ETRAMPs appear to
localize to the liver stage or blood stage parasitophorous vacuole and have been shown
to interact with host proteins. For example, PyUIS3/PY03011 interacts with L-FABP
[13] and PFE1570w, another ETRAMP, interacts with human apolipoproteins [34]. Therefore, this family of proteins may interact with multiple host proteins.

A PyUIS3/PY03011-knockout parasite, Pyuis3(-), can develop into sporozoites and invade
hepatocytes, but can not develop into merozoites, indicating that PyUIS3/PY03011 is
not required for sporozoite development or sporozoite invasion of salivary glands
or hepatocytes, but is required for liver stage development [35]. Mice immunized with Pyuis3(-) knockout parasites are protected against a wild-type
P. yoelii challenge [35]. Therefore, an immune response against PyUIS3/PY03011 is not essential for protection.
However, since PyUIS3/PY03011 is essential for hepatocyte development, it is not surprising
that an immune response against this protein can help protect mice against a P. yoelii challenge.

PFI0580c, the P. falciparum ortholog of PY03424, encodes a putative cysteine protease inhibitor, falstatin [14]. This protein can inhibit the P. falciparum cysteine proteases, falcipain-2 and falcipain-3, as well as other Plasmodium and human cysteine proteases. Western and mass spectrophotometry analyses indicate
that PFI0580c is expressed in sporozoites, as well as the ring, schizont and merozoite
stages of the P. falciparum life-cycle, but not in trophozoites, the stage at which cysteine protease activity
is greatest [14]. Antibodies against falstatin can inhibit merozoite infection of erythrocytes [14]. Therefore, this protein appears to be involved in erythrocyte invasion.

The P. berghei ortholog, P. berghei inhibitor of cysteine proteases (PbICP), has also been well characterized [36]. Similar to PY03424 and PFI0580c, PbICP is expressed in multiple stages of the parasite
life-cycle. In sporozoites, it localizes to micronemes and is secreted by gliding
sporozoites. In infected liver cells, it localizes to the parasitophorous vacuole.
PbICP appears to play an important role in both of these stages. Pre-incubation of
sporozoites with PbICP-specific antibody inhibits sporozoite infection of HepG2 cells.
Therefore, this protein appears to play a role in sporozoite invasion of hepatocytes.
In addition, HepG2 cells transfected with a plasmid expressing PbICP are resistant
to apoptosis-inducing reagents. Therefore, PbICP may inhibit the programmed cell death
of parasite-infected liver cells, perhaps by inhibiting one or more of the cellular
proteases involved in this process. These studies indicate that the PY03424 orthologs,
falstatin and PbICP, play a critical role in multiple stages of the parasite life-cycle,
including sporozoite invasion of hepatocytes, liver stage development and merozoite
infection of erythrocytes.

It is not known what the correlates of protection are in these studies. PyUIS3/PY03011
is expressed in the sporozoite and liver stages [13]. Since PyUIS3-knockout parasites can infect hepatocytes, this protein is not required
for sporozoite infection of hepatocytes [35]. Therefore, antibodies against PY03011/PyUIS3 may not have an impact on sporozoite
infectivity. Since PyUIS3-knockout parasites cannot develop into functional merozoites,
this protein is essential for liver stage development [35]. PyUIS3 localizes to the liver stage parasitophorous vacuole and should not be accessible
to circulating antibodies. Therefore, the protection induced by a PY03011-based vaccine
may be more dependent on a cellular response than a humoral response.

The PY03424 orthologs, falstatin and PbICP, play critical roles in multiple stages
of the parasite life-cycle, including sporozoite infection of hepatocytes, liver stage
development and merozoite infection of erythrocytes. Antibodies against these proteins
can inhibit sporozoite infection of hepatocytes and merozoite infection of erythrocytes
[14,36]. Therefore, PY03424-specific antibodies may have played a critical role in the protection
observed in this study. However, since this protein also appears to be involved in
inhibiting apoptosis of infected hepatocytes, PY03424-specific T cell responses may
have also played a role in protection.

The protection studies reported here were performed in CD1 outbred mice. Although
studies with other malaria antigens have indicated that higher levels of protection
can be attained in inbred mouse strains, protection is often antigen and strain-specific.
For example, a DNA-PyCSP vaccine vector can protect BALB/c (H-2d) mice against a P. yoelii challenge, but cannot protect a high percentage of A/J (H-2a) or B10.BR (H-2k) mice. Conversely, a DNA-PyHEP17 vaccine vector can protect A/J and B10.BR mice,
but cannot protect a high percentage of BALB/c mice [37]. To avoid the possibility of missing potentially protective vaccine antigens due
to HLA-restricted responses, protection studies were performed in CD1 outbred mice.

These results suggest that combining vaccine antigens can have a synergistic impact
on protection. Specifically, vaccine combinations with vectors that express PY03011
and PY03424, or PY03011, PY03424 and PY03661 protected mice at significantly higher
levels than vaccines that express the individual antigens. Other studies have also
shown that combining vaccines can enhance protection, as well as circumvent the HLA-restricted
protection observed with some single antigen vaccines. For example, a combination
vaccine containing two DNA vectors that express PyCSP and PyHEP17 protected a higher
percentage of BALB/c, A/J and B10.BR mice than either the DNA-PyCSP or DNA-PyHEP17
vector alone [37]. In addition, monkeys immunized with DNA and vaccinia vectors expressing four P. knowlesi antigens (PkCSP, PkTRAP, PkAMA1 and PkMSP142) controlled a P. knowlesi challenge significantly better than monkeys immunized with DNA and vaccinia vectors
expressing only PkCSP [38]. Combining vaccines, however, can have several disadvantages. A multi-component vaccine
may be more expensive to manufacture than a vaccine that contains a single component.
In addition, there is a risk that one vaccine component can have an immunosuppressive
effect on the other components. For example, a vaccine containing nine different DNA-P. falciparum vectors elicited significantly lower immune responses against each individual antigen
than a vaccine containing the individual vectors [39]. Therefore, combining vaccine antigens will need to be evaluated empirically to see
if synergistic, additive or antagonistic responses are observed.

Conclusions

The results presented here suggest that characterizing the protective potential of
new malaria vaccine antigens, such as PY03011 and PY03424, may contribute to the development
of an efficacious malaria vaccine that can overcome the antigenic diversity of malaria
parasites. In future studies, these antigens will be tested in combination with other
protective antigens, such as PyCSP, to see if even higher levels of protection can
be achieved.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

KL conceived and designed the experiments. KL, JA, KG, EA and JS performed the experiments.
KL and NP analyzed the data. JA and MS contributed information and reagents. KL, NP
and TR wrote the manuscript. All authors have read and approved the final manuscript.

Acknowledgements

We would like to thank Jessica Bolton, Joyce Wanga, Phuong Thao Pham, Nicole Barnes
and Dianne Litilit for their excellent technical assistance. The views expressed in
this paper are those of the authors and do not necessarily reflect the official policy
or position of the Department of the Navy, Department of Defense, nor the U.S. Government.
This work was supported by work unit number 6000.RAD1.F.A0309. The experiments reported
herein were conducted in compliance with the Animal Welfare Act and in accordance
with the principles set forth in the "Guide for the Care and Use of Laboratory Animals",
Institute of Laboratory Animals Resources, National Research Council, National Academy
Press, 1995. Thomas Richie is a military service member and Kalpana Gowda and Martha
Sedegah are employees of the U.S. Government. This work was prepared as part of their
official duties. Title 17 U.S.C. §105 provides that 'Copyright protection under this
title is not available for any work of the United States Government.' Title 17 U.S.C.
§101 defines U.S. Government work as work prepared by a military service member or
employee of the U.S. Government as part of that person's official duties.